Antibody-displaying extracellular vesicles for targeted cancer therapy.

Extracellular vesicles (EVs) function as natural delivery vectors and mediators of biological signals across tissues. Here, by leveraging these functionalities, we show that EVs decorated with an antibody-binding moiety specific for the fragment crystallizable (Fc) domain can be used as a modular delivery system for targeted cancer therapy. The Fc-EVs can be decorated with different types of immunoglobulin G antibody and thus be targeted to virtually any tissue of interest. Following optimization of the engineered EVs by screening Fc-binding and EV-sorting moieties, we show the targeting of EVs to cancer cells displaying the human epidermal receptor 2 or the programmed-death ligand 1, as well as lower tumour burden and extended survival of mice with subcutaneous melanoma tumours when systemically injected with EVs displaying an antibody for the programmed-death ligand 1 and loaded with the chemotherapeutic doxorubicin. EVs with Fc-binding domains may be adapted to display other Fc-fused proteins, bispecific antibodies and antibody-drug conjugates.

Tumor-associated NK cells drive MDSC-mediated tumor immune tolerance through the IL-6/STAT3 axis.

Apart from their killer identity, natural killer (NK) cells have integral roles in shaping the tumor microenvironment. Through immune gene deconvolution, the present study revealed an interplay between NK cells and myeloid-derived suppressor cells (MDSCs) in nonresponders of immune checkpoint therapy. Given that the mechanisms governing the outcome of NK cell-to-myeloid cell interactions remain largely unknown, we sought to investigate the cross-talk between NK cells and suppressive myeloid cells. Upon contact with tumor-experienced NK cells, monocytes and neutrophils displayed increased expression of MDSC-related suppressive factors along with increased capacities to suppress T cells. These changes were accompanied by impaired antigen presentation by monocytes and increased ER stress response by neutrophils. In a cohort of patients with sarcoma and breast cancer, the production of interleukin-6 (IL-6) by tumor-infiltrating NK cells correlated with S100A8/9 and arginase-1 expression by MDSCs. At the same time, NK cell-derived IL-6 was associated with tumors with higher major histocompatibility complex class I expression, which we further validated with b2m-knockout (KO) tumor mice models. Similarly in syngeneic wild-type and IL-6 KO mouse models, we then demonstrated that the accumulation of MDSCs was influenced by the presence of such regulatory NK cells. Inhibition of the IL-6/signal transducer and activator of transcription 3 (STAT3) axis alleviated suppression of T cell responses, resulting in reduced tumor growth and metastatic dissemination. Together, these results characterize a critical NK cell-mediated mechanism that drives the development of MDSCs during tumor immune escape.

Natural killer cells drive 4-1BBL positive uveal melanoma towards EMT and metastatic disease.

BACKGROUND: Inflammation in the eye is often associated with aggravated ocular diseases such as uveal melanoma (UM). Poor prognosis of UM is generally associated with high potential of metastatic liver dissemination. A strong driver of metastatic dissemination is the activation of the epithelial-mesenchymal transition (EMT) regulating transcription factor ZEB1, and high expression of ZEB1 is associated with aggressiveness of UM. While ZEB1 expression can be also associated with immune tolerance, the underlying drivers of ZEB1 activation remain unclear. METHODS: Transcriptomic, in vitro, ex vivo, and in vivo analyses were used to investigate the impact on clinical prognosis of immune infiltration in the ocular tumor microenvironment. A metastatic liver dissemination model of was developed to address the role of natural killer (NK) cells in driving the migration of UM. RESULTS: In a pan-cancer TCGA analysis, natural killer (NK) cells were associated with worse overall survival in uveal melanoma and more abundant in high-risk monosomy 3 tumors. Furthermore, uveal melanoma expressed high levels of the tumor necrosis factor superfamily member 4-1BB ligand, particularly in tumors with monosomy 3 and BAP1 mutations. Tumors expressing 4-1BB ligand induced CD73 expression on NK cells accompanied with the ability to promote tumor dissemination. Through ligation of 4-1BB, NK cells induced the expression of the ZEB1 transcription factor, leading to the formation of liver metastasis of uveal melanoma. CONCLUSIONS: Taken together, the present study demonstrates a role of NK cells in the aggravation of uveal melanoma towards metastatic disease.

Minimal residual disease status is the prognostic determinant following high-dose treatment for patients with multiple myeloma.

BACKGROUND: The presence of minimal residual disease (MRD+) following autologous stem cell transplantation (ASCT) in multiple myeloma represents a poor prognostic factor for progression-free survival (PFS) and overall survival (OS). METHODS: At our department, we recommend lenalidomide maintenance for patients who are MRD+ after ASCT, while MRD-negative (MRD-) patients, after information about the national guidelines, were not advised to follow this regimen. RESULTS: Out of the total 228 patients, 175 received ASCT following first-line induction (MRD- 92 (53%), MRD+ 83 (47%), at 2 months post-ASCT), while 53 underwent ASCT after second-line treatment (MRD- 27 (51%), MRD+ 26 (49%), at the same time point). Comparatively, MRD- patients who did not receive maintenance demonstrated better OS than MRD+ patients who received upfront ASCT and maintenance treatment (96% vs. 86%, p = 0.030, at 3 years). However, nonsignificant difference was found in PFS (76% vs. 62%, at 3 years). Furthermore, second-line ASCT, MRD- non-maintained patients exhibited significantly better PFS than MRD+ (71% vs. 27%, p > 0.001, at 3 years). However, OS was better but nonsignificant (96% vs. 76%, at 3 years). Fluorescence in situ hybridization (FISH) analysis was performed on 141 out of the 228 patients. Of these, 85 (60%) patients were deemed standard risk (SR), and 56 (40%) were classified as high risk (HR). In the SR cohort, MRD- patients exhibited better PFS and OS than MRD+ patients (71% vs. 59% and 100% vs. 85%, respectively). In the HR cohort, the MRD- patients showed superior PFS but similar OS compared to MRD+ patients (66% vs. 42% and 81% vs. 80%, respectively). CONCLUSIONS: Our results indicate that being MRD- is a more crucial prognostic factor for the 3-year PFS and OS than the presence of high-risk cytogenetic markers or undergoing maintenance treatment. The latter appears insufficient, particularly for MRD+ patients following ASCT in the second-line setting, suggesting that these patients may require a more intensive treatment approach.

Modulation of lytic molecules restrain serial killing in γδ T lymphocytes.

γδ T cells play a pivotal role in protection against various types of infections and tumours, from early childhood on and throughout life. They consist of several subsets characterised by adaptive and innate-like functions, with Vγ9Vδ2 being the largest subset in human peripheral blood. Although these cells show signs of cytotoxicity, their modus operandi remains poorly understood. Here we explore, using live single-cell imaging, the cytotoxic functions of γδ T cells upon interactions with tumour target cells with high temporal and spatial resolution. While γδ T cell killing is dominated by degranulation, the availability of lytic molecules appears tightly regulated in time and space. In particular, the limited co-occurrence of granzyme B and perforin restrains serial killing of tumour cells by γδ T cells. Thus, our data provide new insights into the cytotoxic arsenal and functions of γδ T cells, which may guide the development of more efficient γδ T cell based adoptive immunotherapies.

A tractable microscopy- and flow cytometry-based method to measure natural killer cell-mediated killing and infiltration of tumor spheroids.

Understanding the anti-tumor activity of immune cells and testing cancer immunotherapies requires conditions that are as life-like as possible. The tumor microenvironment (TME) describes a complex sum of cellular and acellular actors that influence both immune cells and tumor cells as well as their interplay. Yet in development phases of new immunotherapies, the screening of drugs and adoptive cell products benefits from reproducible and controlled conditions. Two-dimensional (2D) cell cultures cannot simultaneously meet these two challenges therefore lacking considerably predictive power owing to their artificial nature. Various 3D tumor models have therefore been implemented to mimic the architecture and intrinsic heterogeneity of a microtumor. This protocol provides an easy-to-follow, time-efficient, material-limited method for live cell killing and infiltration of single tumor spheroids. It uses multicellular tumor spheroids grown scaffold-free and allows co-culture with immune cells. This protocol is optimized for natural killer (NK) cell functionality assays. However, it can be transferred to other immune cells, in particular cytotoxic T cells. This assay can be analysed using life cell imaging (here with the IncuCyte S3 system) and/or flow cytometry.

Accumulation of tissue-resident natural killer cells, innate lymphoid cells, and CD8+ T cells towards the center of human lung tumors.

Lung cancer is a leading cause of cancer-related death worldwide. Despite recent advances in tissue immunology, little is known about the spatial distribution of tissue-resident lymphocyte subsets in lung tumors. Using high-parameter flow cytometry, we identified an accumulation of tissue-resident lymphocytes including tissue-resident NK (trNK) cells and CD8+ tissue-resident memory T (TRM) cells toward the center of human non-small cell lung carcinomas (NSCLC). Chemokine receptor expression patterns indicated different modes of tumor-infiltration and/or residency between trNK cells and CD8+ TRM cells. In contrast to CD8+ TRM cells, trNK cells and ILCs generally expressed low levels of immune checkpoint receptors independent of location in the tumor. Additionally, granzyme expression in trNK cells and CD8+ TRM cells was highest in the tumor center, and intratumoral CD49a+CD16- NK cells were functional and responded stronger to target cell stimulation than their CD49a- counterparts, indicating functional relevance of trNK cells in lung tumors. In summary, the present spatial mapping of lymphocyte subsets in human NSCLC provides novel insights into the composition and functionality of tissue-resident immune cells, suggesting a role for trNK cells and CD8+ TRM cells in lung tumors and their potential relevance for future therapeutic approaches.

Challenges in αCD38-chimeric antigen receptor (CAR)-expressing natural killer (NK) cell-based immunotherapy in multiple myeloma: Harnessing the CD38dim phenotype of cytokine-stimulated NK cells as a strategy to prevent fratricide.

BACKGROUND AIMS: Adoptive cell therapy with chimeric antigen receptor (CAR)-expressing natural killer (NK) cells is an emerging approach that holds promise in multiple myeloma (MM). However, the generation of CAR-NK cells targeting CD38 is met with obstacles due to the expression of CD38 on NK cells. Knock-out of CD38 is currently explored as a strategy, although the consequences of the lack of CD38 expression with regards to engraftment and activity in the bone marrow microenvironment are not fully elucidated. Here, we present an alternative approach by harnessing the CD38dim phenotype occurring during long-term cytokine stimulation of primary NK cells. METHODS: Primary NK cells were expanded from peripheral blood mononuclear cells by long-term IL-2 stimulation. During expansion, the CD38 expression was monitored in order to identify a time point when introduction of a novel affinity-optimized αCD38-CAR confered optimal viability, i.e. prevented fratricide. CD38dim NK cells were trasduced with retroviral vectors encoding for the CAR trasngene and their functionality was assessed in in vitro activation and cytotoxicity assays. RESULTS: We verified the functionality of the αCD38-CAR-NK cells against CD38+ cell lines and primary MM cells. Importantly, we demonstrated that αCD38-CAR-NK cells derived from patients with MM have increased activity against autologous MM samples ex vivo. CONCLUSIONS: Overall, our results highlight that incorporation of a functional αCD38-CAR construct into a suitable NK-cell expansion and activation protocol results in a potent and feasible immunotherapeutic strategy for the treatment of patients with MM.

SHP-1 localization to the activating immune synapse promotes NK cell tolerance in MHC class I deficiency.

Natural killer (NK) cells recognize virally infected cells and tumors. NK cell function depends on balanced signaling from activating receptors, recognizing products from tumors or viruses, and inhibitory receptors (such as KIR/Ly49), which recognize major histocompatibility complex class I (MHC-I) molecules. KIR/Ly49 signaling preserves tolerance to self but also conveys reactivity toward MHC-I-low target cells in a process known as NK cell education. Here, we found that NK cell tolerance and education were determined by the subcellular localization of the tyrosine phosphatase SHP-1. In mice lacking MHC-I molecules, uneducated, self-tolerant Ly49A+ NK cells showed accumulation of SHP-1 in the activating immune synapse, where it colocalized with F-actin and the signaling adaptor protein SLP-76. Education of Ly49A+ NK cells by the MHC-I molecule H2Dd led to reduced synaptic accumulation of SHP-1, accompanied by augmented signaling from activating receptors. Education was also linked to reduced transcription of Ptpn6, which encodes SHP-1. Moreover, synaptic SHP-1 accumulation was reduced in NK cells carrying the H2Dd-educated receptor Ly49G2 but not in those carrying the noneducating receptor Ly49I. Colocalization of Ly49A and SHP-1 outside of the synapse was more frequent in educated compared with uneducated NK cells, suggesting a role for Ly49A in preventing synaptic SHP-1 accumulation in NK cell education. Thus, distinct patterning of SHP-1 in the activating NK cell synapse may determine NK cell tolerance.

Generation of NK cells with chimeric-switch receptors to overcome PD1-mediated inhibition in cancer immunotherapy.

Multiple myeloma (MM) is an incurable hematological cancer, in which immune checkpoint inhibition (ICI) with monoclonal antibodies (mAbs) has failed due to uncontrollable immune responses in combination therapies and lack of efficacy in monotherapies. Although NK cell-specific checkpoint targets such as NKG2A and KIRs are currently being evaluated in clinical trials, the clinical impact of NK cells on the PD1 cascade is less well understood compared to T cells. Furthermore, while NK cells have effector activity within the TME, under continuous ligand exposure, NK cell dysfunctionality may occur due to interaction of PD1 and its ligand PD-L1. Due to above-mentioned factors, we designed novel NK cell specific PD1-based chimeric switch receptors (PD1-CSR) by employing signaling domains of DAP10, DAP12 and CD3ζ to revert NK cell inhibition and retarget ICI. PD1-CSR modified NK cells showed increased degranulation, cytokine secretion and cytotoxicity upon recognition of PD-L1+ target cells. Additionally, PD1-CSR+ NK cells infiltrated and killed tumor spheroids. While primary NK cells (pNK), expressing native PD1, showed decreased degranulation and cytokine production against PD-L1+ target cells by twofold, PD1-CSR+ pNK cells demonstrated increased activity upon PD-L1+ target cell recognition and enhanced antibody-dependent cellular cytotoxicity. PD1-CSR+ pNK cells from patients with MM increased degranulation and cytokine expression against autologous CD138+PD-L1+ malignant plasma cells. Taken together, the present results demonstrate that PD1-CSR+ NK cells enhance and sustain potent anti-tumor activity in a PD-L1+ microenvironment and thus represent a promising strategy to advance adoptive NK cell-based immunotherapies toward PD-L1+ cancers.

Adaptive single-KIR+NKG2C+ NK cells expanded from select superdonors show potent missing-self reactivity and efficiently control HLA-mismatched acute myeloid leukemia.

BACKGROUND: Natural killer (NK) cells hold great promise as a source for allogeneic cell therapy against hematological malignancies, including acute myeloid leukemia (AML). Current treatments are hampered by variability in NK cell subset responses, a limitation which could be circumvented by specific expansion of highly potent single killer immunoglobulin-like receptor (KIR)+NKG2C+ adaptive NK cells to maximize missing-self reactivity. METHODS: We developed a GMP-compliant protocol to expand adaptive NK cells from cryopreserved cells derived from select third-party superdonors, that is, donors harboring large adaptive NK cell subsets with desired KIR specificities at baseline. We studied the adaptive state of the cell product (ADAPT-NK) by flow cytometry and mass cytometry as well as cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq). We investigated the functional responses of ADAPT-NK cells against a wide range of tumor target cell lines and primary AML samples using flow cytometry and IncuCyte as well as in a mouse model of AML. RESULTS: ADAPT-NK cells were >90% pure with a homogeneous expression of a single self-HLA specific KIR and expanded a median of 470-fold. The ADAPT-NK cells largely retained their adaptive transcriptional signature with activation of effector programs without signs of exhaustion. ADAPT-NK cells showed high degranulation capacity and efficient killing of HLA-C/KIR mismatched tumor cell lines as well as primary leukemic blasts from AML patients. Finally, the expanded adaptive NK cells had preserved robust antibody-dependent cellular cytotoxicity potential and combination of ADAPT-NK cells with an anti-CD16/IL-15/anti-CD33 tri-specific engager led to near-complete killing of resistant CD45dim blast subtypes. CONCLUSIONS: These preclinical data demonstrate the feasibility of off-the-shelf therapy with a non-engineered, yet highly specific, NK cell population with full missing-self recognition capability.

Tumor MHC class I expression alters cancer-associated myelopoiesis driven by host NK cells.

Downregulation of MHC class I (MHCI) molecules on tumor cells is recognized as a resistance mechanism of cancer immunotherapy. Given that MHCI molecules are potent regulators of immune responses, we postulated that the expression of MHCI by tumor cells influences systemic immune responses. Accordingly, mice-bearing MHCI-deficient tumor cells showed reduced tumor-associated extramedullary myelopoiesis in the spleen. Depletion of natural killer (NK) cells abrogated these differences, suggesting an integral role of immune-regulatory NK cells during tumor progression. Cytokine-profiling revealed an upregulation of TNF-α by NK cells in tumors and spleen in mice-bearing MHCI expressing tumors, and inhibition of TNF-α enhanced host myelopoiesis in mice receiving adoptive transfer of tumor-experienced NK cells. Our study highlights a critical role of NK cells beyond its identity as a killer lymphocyte and more importantly, the potential host responses to a localized tumor as determined by its MHCI expression.

Conditioning with melphalan 200 mg/m2 and subsequent ASCT improves progression-free and overall survival in elderly myeloma patients compared to standard of care.

OBJECTIVES: Despite the effectiveness of newer drugs for the treatment of multiple myeloma (MM), the outcomes are further improved by subsequent autologous stem cell transplantation (ASCT). Data on effectiveness in older patients are limited. We compared outcomes in patients aged 65-75 years depending on whether they were treated with ASCT or not and compared those to outcomes in patients <65 years. METHODS: This was a retrospective, single-center study. We compared progression-free survival (PFS) and overall survival (OS) for all MM patients below and above the age of 65 years treated ± ASCT at the Karolinska University Hospital between 2010 and 2020. PFS and OS were calculated by the Kaplan-Meier method. Variables affecting PFS and OS were evaluated using Cox regression model. RESULTS: Both PFS and OS were improved in the group 65-75 years treated +ASCT compared to those treated pharmacologically (p = 0.008 and p < 0.001, respectively). There were no significant differences between patients <65 years and those 65-75 years treated with ASCT. CONCLUSION: The findings indicate that even patients >65 years should be evaluated as candidates for ASCT. An individualized approach supported by a frailty/geriatric assessment score could assist clinicians to select the appropriate treatment for each patient.

Assessment of T Cell Receptor Complex Expression Kinetics in Natural Killer Cells.

Among the polypeptides that comprise the T cell receptor (TCR), only CD3ζ is found in Natural Killer (NK) cells, where it transmits signals from activating receptors such as CD16 and NKp46. NK cells are potent immune cells that recognize target cells through germline-encoded activating and inhibitory receptors. Genetic engineering of NK cells enables tumor-specific antigen recognition and, thus, has a significant promise in adoptive cell therapy. Ectopic expression of engineered TCR components in T cells leads to mispairing with the endogenous components, making a knockout of the endogenous TCR necessary. To circumvent the mispairing of TCRs or the need for knockout technologies, TCR complex expression has been studied in NK cells. In the current study, we explored the cellular processing of the TCR complex in NK cells. We observed that in the absence of CD3 subunits, the TCR was not expressed on the surface of NK cells and vice versa. Moreover, a progressive increase in surface expression of TCR between day three and day seven was observed after transduction. Interestingly, the TCR complex expression in NK92 cells was enhanced with a proteasome inhibitor (bortezomib) but not a lysosomal inhibitor (chloroquine). Additionally, we observed that the TCR complex was functional in NK92 cells as measured by estimating CD107a as a degranulation marker, IFNγ cytokine production, and killing assays. NK92 cells strongly degranulated when CD3ε was engaged in the presence of TCR, but not when only CD3 was overexpressed. Therefore, our findings encourage further investigation to unravel the mechanisms that prevent the surface expression of the TCR complex.

Engineered NK Cells Against Cancer and Their Potential Applications Beyond.

Cell therapy is an innovative therapeutic concept where viable cells are implanted, infused, or grafted into a patient to treat impaired or malignant tissues. The term was first introduced circa the 19th century and has since resulted in multiple breakthroughs in different fields of medicine, such as neurology, cardiology, and oncology. Lately, cell and gene therapy are merging to provide cell products with additional or enhanced properties. In this context, adoptive transfer of genetically modified cytotoxic lymphocytes has emerged as a novel treatment option for cancer patients. To this day, five cell therapy products have been FDA approved, four of which for CD19-positive malignancies and one for B-cell maturation antigen (BCMA)-positive malignancies. These are personalized immunotherapies where patient T cells are engineered to express chimeric antigen receptors (CARs) with the aim to redirect the cells against tumor-specific antigens. CAR-T cell therapies show impressive objective response rates in clinical trials that, in certain instances, may reach up to 80%. However, the life-threatening side effects associated with T cell toxicity and the manufacturing difficulties of developing personalized therapies hamper their widespread use. Recent literature suggests that Natural Killer (NK) cells, may provide a safer alternative and an 'off-the-shelf' treatment option thanks to their potent antitumor properties and relatively short lifespan. Here, we will discuss the potential of NK cells in CAR-based therapies focusing on the applications of CAR-NK cells in cancer therapy and beyond.

Autologous NK cells as consolidation therapy following stem cell transplantation in multiple myeloma.

Few approaches have been made toward exploring autologous NK cells in settings of cancer immunotherapy. Here, we demonstrate the feasibility of infusing multiple doses of ex vivo activated and expanded autologous NK cells in patients with multiple myeloma (MM) post-autologous stem cell transplantation. Infused NK cells were detected in circulation up to 4 weeks after the last infusion. Elevations in plasma granzyme B levels were observed following each consecutive NK cell infusion. Moreover, increased granzyme B levels were detected in bone marrow 4 weeks after the last infusion. All measurable patients had objective, detectable responses after NK cell infusions in terms of reduction in M-component and/or minimal residual disease. The present study demonstrates that autologous NK cell-based immunotherapy is feasible in a setting of MM consolidation therapy. It opens up the possibility for usage of autologous NK cells in clinical settings where patients are not readily eligible for allogeneic NK cell-based immunotherapies.

NK cell frequencies, function and correlates to vaccine outcome in BNT162b2 mRNA anti-SARS-CoV-2 vaccinated healthy and immunocompromised individuals.

Adaptive immune responses have been studied extensively in the course of mRNA vaccination against COVID-19. Considerably fewer studies have assessed the effects on innate immune cells. Here, we characterized NK cells in healthy individuals and immunocompromised patients in the course of an anti-SARS-CoV-2 BNT162b2 mRNA prospective, open-label clinical vaccine trial. See trial registration description in notes. Results revealed preserved NK cell numbers, frequencies, subsets, phenotypes, and function as assessed through consecutive peripheral blood samplings at 0, 10, 21, and 35 days following vaccination. A positive correlation was observed between the frequency of NKG2C+ NK cells at baseline (Day 0) and anti-SARS-CoV-2 Ab titers following BNT162b2 mRNA vaccination at Day 35. The present results provide basic insights in regards to NK cells in the context of mRNA vaccination, and have relevance for future mRNA-based vaccinations against COVID-19, other viral infections, and cancer.Trial registration: The current study is based on clinical material from the COVAXID open-label, non-randomized prospective clinical trial registered at EudraCT and clinicaltrials.gov (no. 2021-000175-37). Description: https://clinicaltrials.gov/ct2/show/NCT04780659?term=2021-000175-37&draw=2&rank=1 .